MXPA05012298A - Process for manufacture of nematode-extracted anticoagulant protein (nap). - Google Patents

Abstract

The present invention provides a process for manufacture of purified Nematode-extracted Anticoagulant Proteins (NAPS), wherein the NAP manufactured by the claimed process method is a NAP drug substance that can be formulated as a NAP drug product. The present invention provides NAP drug substances and NAP drug products manufactured by the process disclosed herein. In one embodiment, the present invention provides a process for manufacture of rNAPc2/proline drug substance and rNAPc2/proline drug product, and provides rNAPc2/proline drug substance manufactured by the process disclosed herein.

Description

PROCESS OF MANUFACTURING AN ANTICOAGULANT PROTEIN EXTRACTED FROM NEMATOTICS (NAP) RELATED APPLICATIONS This application claims the priority benefit of the pending application "Method of Treatment of Hemorrhagic Disease Using a Vlla Factor Factor / Tissue Factor Inhibitor" filed on May 6, 2003. Field of the Invention The present invention relates to a process for the manufacture of proteins that are anticoagulants in human plasma and proteins produced by this process. Specifically, the present invention relates to processes for the manufacture of Anticoagulant Proteins Extracted from Nematodes (NAPs) and related to the NAPs manufactured by this process. In particular, the present invention relates to NAP drug substances and NAP drug products and processes for their manufacture. Background of the Invention The discovery and purification of therapeutic proteins that have potential value as pharmaceuticals, can be carried out in a research laboratory using materials and methods that are not suitable for the large-scale commercial production of pharmaceutical products. To generate pharmaceutical products on a commercial scale, biotechnology manufacturing operations must be robust and scalable without compromising product quality (Gottschalk, 2003, BioProcess Intl 1 (4): pages 54 to 61). Manufacturing processes for pharmaceutical products must provide cost-effective methods, improve product yields, have sufficient capacity to meet demand, and ideally must provide the scaling capacity of the process to respond to fluctuations in demand. . Manufacturing processes for therapeutic proteins must develop cost-effective methods for the production of large amounts of the protein in a functional form, as well as methods for purifying the protein to generate a pharmaceutical product of a purity suitable for its intended use. "Protein scale" research methods of purification, also known as "laboratory scale" methods, or "bench scale", are often closely linked to the methods that were used to discover and characterize the therapeutic protein. Often, a yield of only micrograms or milligrams of purified protein is sufficient to characterize and engineer the protein sequence. Even after an expression system for recombinantly producing a therapeutic protein has been developed, such expression systems are not necessarily suitable for producing the protein on a commercial scale. In addition, purification scale purification methods can utilize organic solvents, strong acids and other reagents that are undesirable or practical on a commercial scale and sometimes not allowed in the manufacture of pharmaceutical products. In addition, these purification methods can use separation methods such as molecular casting or high performance liquid chromatography (HPLC) which are powerful purification methods in the laboratory, but can not easily scale at commercial production levels. Pilot-scale processes, eg, fermentation volumes of 10L to 100L in a host cell expressing the therapeutic protein, are suitable for further studying the production process or for producing sufficient quantities of a therapeutic protein for early clinical studies, but Even pilot scale processes can not always be scaled to manufacture the quantities required for the clinical studies of the final phase. One method to increase biotechnology manufacturing capacity is to extend the production capacity and efficiency of microbial expression systems. A variety of well-established biological "factories" are available for the production of therapeutic proteins. Nevertheless, because the production of the functional protein is intimately related to the cellular machinery of the organism that produces the protein, each expression system has advantages and disadvantages to be used in a large-scale production of pharmaceutical products, depending on the protein. E. coli has been the "factory" of choice for the expression of many proteins because it is easy to handle, grows rapidly, requires growth media that are not expensive and can secrete proteins in the medium, which facilitates recovery . However, many eukaryotic proteins produced in E. coli are produced in an unfinished and non-functional form that lacks glycosylation and other post-translational modifications, as well as the formation of proteins with an appropriate disulfide bond and a fold three-dimensional. In addition, the material produced in E. coli may have endotoxin contamination. Similar restrictions are often found using Bacillus species as expression systems. Mammalian cell culture systems provide small amounts of eukaryotic proteins with appropriate glycosylation and folding, but mammalian cell cultures are expensive, can be difficult to scale to commercial production levels and can be unstable and require the use of animal serum. Insect cell expression systems are rapid, and relatively easy to develop, and offer good expression levels of mammalian proteins, but can be expensive, can only be scaled moderately, and can produce inappropriate glycosylation. Yeast expression systems are popular because they are easy to grow, fast and can be scaled; however, some yeast expression systems have produced inconsistent results, and sometimes it is difficult to achieve high yields. A system of yeast expression that has shown great promise is the methanotrophic Pichia pastoris. Compared with other eukaryotic expression systems, Pichia offers many advantages, because it does not have the problem of endotoxins associated with the bacterium, nor the problems of viral contamination of proteins produced in animal cell cultures (Ciño, Am Biotech Lab, May 1999). Pichia uses methanol as the carbon source in the absence of glucose, using an alcohol oxidase promoter induced by methanol (AOX1), which generally controls the expression of the enzyme which catalyzes the first step in methanol metabolism, as a promoter that can be induced by methanol to direct the expression of heterologous proteins. The prolific growth rate of the Pichia makes it easily scalable to commercial scale production, although the escalation challenges include pH control, oxygen limitation, nutrient limitation, temperature fluctuation and safety considerations for the use of methanol ( Gottschalk, 2003, BioProcess Intl 1 (4): pages 54 to 61; Ciño Am Biotech Lab, May 1999). Production under the conditions of Good Manufacturing Practice (cGMP) is possible with Pichia pastoris, on a scale of 1000L fermentations (Gottschalk, 2003, BioProcess Intl 1 (4): pages 54 to 61). Another method to increase the capacity of biotechnological manufacturing is to improve protein recovery and downstream processing of fermentation products. In downstream processing, processes must be adjustable to accommodate changes and improvements in fermentation titration, media composition and cell viability, while maximizing the productivity of existing capacity (Gottschalk, 2003, BioProcess Intl 1 (4): pages 54 to 61). Recent advances in chromatography and filtration provide significant increases in selectivity, recovery and offer high capacities and low cycle times to be compatible with large volumes and high expression levels of current batch feed fermentation processes (Gottschalk, 2003 , BioProcess Intl 1 (4): pages 54 to 61). Despite the great advantages for the improvement of biotechnological manufacturing, there are no global solutions for each protein. The manufacturing process for a specific therapeutic protein requires novel and innovative solutions to problems that may be specific to that protein or family of proteins. In a similar way, successful commercial applications often depend on a combination of specific properties of the protein or protein family and the production processes used to make the protein or protein family as pharmaceuticals. Summary of the Invention The present invention provides a process for the manufacture of purified anticoagulant proteins extracted from nematodes (NAPs), and substances for purified NAP drugs and NAP drug products manufactured by this process. The present invention provides a process for the manufacture of large quantities (commercial scale) of NAP drug substances and NAP drug products. In particular, the present invention provides a process for the manufacture of a NAP drug substance that includes the steps of: (a) a fermentation process comprising producing the NAP in a suitable host, wherein at least one sequence is integrated which encodes the NAP in the host genome; (b) a recovery process in which the NAP is separated from the cell and cell debris; and (c) a purification process for purifying the NAP drug substance from contaminants. A suitable host is Pichia pastoris. The process may also include the introduction of the drug substance NAP into a final formulation of a drug. The process may also include a replenishment process that includes bulk filtration of the NAP drug substance in the final drug formulation, and a filling process which may include the supply of the drug substance NAP in the final formulation of the drug in dosage forms to generate the NAP drug product and may further include the lyophilization of the drug product NAP. The process provided herein can be used to manufacture the purified NAP drug substance or the NAP drug product from rNAPc2 (AcaNAPc2), rNAPc2 / proline (AcaNAPc2 / proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44, or AcaNAP46. The present invention provides a NAP drug substance manufactured by the process described herein. The present invention provides a NAP drug substance manufactured using a NAP selected from, but not limited to, rNAPc2 (AcaNAPc2), rNAPc2 / proline (AcaNAPc2 / proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5 , AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44, or AcaNAP46. In one embodiment, a NAP drug substance of the present invention can be manufactured using rNAPc2 / proline. The present invention further provides a NAP drug product manufactured by the process described herein. The present invention provides a NAP drug product manufactured using a NAP selected from, but not limited to, rNAPc2 (AcaNAPc2), rN APc2 / proline (AcaNAPc2 / proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44, or AcaNAP46. In one embodiment, a NAP drug substance of the present invention is manufactured using rNAPc2 / proline. According to another aspect, the present invention provides a process for the manufacture of drug substances rN APc2 / proinin and drug products r APc2 / prolin. The present invention further provides an rNAPc2 / proline drug substance and rNAPc2 / proline drug products manufactured by the process described herein. In particular, the present invention provides a process for the manufacture of an rNAPc2 / proline drug substance that includes a fermentation process, a recovery process and a purification process. The process provided here includes a fermentation process in which rNAPc2 / proline is produced in Pichia pastoris that has at least one sequence encoding the r APc2 / proline that is integrated into the genome, where the fermentation process includes a fermentation of seed to cultivate the host cell in a desired cell density and a production fermentation process comprising glycerol batch fermentation, glycerol fed batch fermentation, methanol adaptation fermentation and methanol induction fermentation, up to about seven days. The process provided herein also includes a recovery process that includes expanded bed ion exchange chromatography to separate rNAPc2 / proline from cells and cell debris. The process provided herein also provides a purification process that includes hydrophobic interaction chromatography using hydrophobic interaction chromatography media, collecting fractions of rNAPc2 / proline at least one ultrafiltration / diafiltration (UF / DF) of the fractions of rNAPc2 / proline. , ion exchange chromatography, and collection of the rNAPc2 / proline fractions from ion exchange chromatography, wherein the rNAPc2 / proline fractions from ion exchange chromatography contain the drug substance rNAPc2 / proline. According to one aspect, the process includes controlling the temperature for fermentation, keeping in particular the temperature of the methanol adaptation fermentation in a range of about 28 ± 2 ° C for about the first four hours and about 25 + 1 ° C for the rest of the methanol adaptation fermentation. According to another aspect, the pH is maintained at a pH of approximately 2.9 ± 0.1 during the methanol adaptation fermentation, and the methanol induction fermentation. In one embodiment, the recovery process includes expanded bed ion exchange chromatography of Streamline SP XL resin at a pH of about 3.2 ± 0.2 and the purification step includes the 15PHE hydrophobic interaction chromatography at a pH of about 3.0 ± 0.1. and Source 15Q ion chromatography, followed by UF / DF of the NAP fractions of ion exchange chromatography. In addition, a process is provided for the manufacture of a rNAPc2 / proline drug liquid product that includes the manufacture of the rNAPc2 / proline drug substance by the process described herein, followed by the introduction of the drug substance rNAPc2 / proline into the drug. final formulation of the drug, a filling process that includes bulk filtration and a filling step comprising the supply of rNAPc2 / proline in a final dosage form, said filling is done in a container or bottle to generate a drug product rNAPc2 / proline liquid, and may further include lyophilization of the drug product rNAPc2 / proline. The present invention provides a rNAPc2 / proline drug liquid product manufactured by this process, and the lyophilized rNAPc2 / proline drug product manufactured by this process. The present invention provides a process for the manufacture of large quantities (commercial scale) of the drug substance NAP, in particular the drug substance rNAPc2 / proline. The substance of the NAP drug manufactured by the process provided herein can be formulated and supplied as a NAP drug product, including a liquid NAP drug product or a lyophilized NAP drug product. Also, the substance of the rNAPc2 / proline drug manufactured by the process provided herein can be formulated and supplied as an rNAPc2 / proline drug product, including a rNAPc2 / proline liquid drug product or a lyophilized rNAPc2 / proline drug product. This process is suitable for efficient production on a commercial scale of the drug substance NAP and the NAP drug products that have desired levels of activity and purity. In contrast, the previously described methods for the purification of the NAPs were research scale methods that could not be scaled for large-scale production of NAPs, and reagents and materials that are undesirable in the production of NAPs were used. drugs and drug products. For example, a recovery process described above consisted of centrifugation to remove the cells. In the above process, the supernatant was then purified by cation exchange chromatography, gel filtration chromatography (also known as molecular casting), and finally, reverse phase chromatography. However, as provided herein, the properties of the NAPs, particularly the rNAPc2 / proline made possible modifications in the process at the research scale to replace the centrifugation step by a method that can be scaled and cleaned, to eliminate both the step of difficult-to-scale gel filtration chromatography, and high-pressure reverse phase liquid chromatography (P-HPLC) comprising the use of a flamable organic solvent and specialized equipment and improving the purity of the final product. In the process provided herein, an expanded bed ion exchange chromatography step, in particular, an expanded bed chromatography step Streamline SP XL, eliminated the multiple unit operations generally used for a recovery step of the commercial process (e.g. , a combination of microfiltration and ultrafiltration). As provided herein, the Streamline SP XL step was used to separate the rNAPc2 / proline from cell debris and the exchange of the product in a regulator suitable for the first purification step. Although the method described above used gel filtration and reverse phase chromatography, these column steps were replaced in the present invention by hydrophobic interaction chromatography (HIC) and anion exchange chromatography, in particular HIC using Source 15PHE and anion exchange using Source 15Q, which resulted in a significant purification of the rNAPc2 / proline through the removal of protein and contaminants that are not proteinaceous. It seems that the relatively low pl of rNAPc2 / proline (pl = 4.1) and other NAPs could be involved in producing the surprising result that the superior bond to the matrix and a higher overall recovery of the HIC step product, depend on carrying out this step of chromatography at a low pH of about 3.2. In addition, the efficiencies of the process resulted from carrying out the steps at a pH of approximately 3, starting from the last fermentation steps through Streamiine chromatography and HIC, which eliminated the exchange of regulator between the steps. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates a vector map of the expression vector rNAPc2 / proline of Pichia pastoris, pYAM7sp8 / rNAPc2 / proline used for the production of rNAPc2 / proline, which shows the reference points. Figures 2A and 2B illustrate a fermentation flow chart showing the materials and reagents used, the process and equipment used, and the conditions that are controlled and monitored for each step of the fermentation process; the fermentation begins with the preparation of the seed flask and continues from the fermentation of seeds through the production fermentation. Figure 3 illustrates a recovery flow diagram showing the materials and reagents used, the process and equipment used, and the conditions that are controlled and monitored during the recovery process. Figures 4A and 4B illustrate a purification flow diagram showing the materials and reagents used, the process and equipment used, and the conditions that are monitored and monitored during the purification process; the purification includes the steps of exchange chromatography and hydrophobic interaction in Source 15PHE, the ultrafiltration / diafiltration step # 1 (UF / DF # 1), ion exchange chromatography Source 15Q, the final UF / DF step, the filtration to bulk, filling and storage of the purified product. Figure 5 illustrates a flow chart of a liquid drug product showing the materials and reagents used, the process and equipment used, and the conditions that are controlled and monitored during the process of making the liquid drug product. This process includes a composition step, a filtration and filling step, and the transfer of flasks with the liquid drug product in storage. Figure 6 illustrates a flow diagram of the lyophilized drug product formulation showing the materials and reagents used, the process and equipment used, and the conditions that are monitored and monitored during the formulation process of the lyophilized drug product. The process includes a UF / DF step, a composition step, a bulk filtration step and refilling, and transfer to the lyophilization unit. Detailed Description of the Invention The present invention provides a process for the manufacture of purified anticoagulant proteins extracted from Nematodes (NAPs), such as those described in US Pat. Nos. 5,863,894; 5,864,009; 5,866,542; 5,866,543; 5,872,098; and 5,945,275 (the total content of each of which is incorporated herein by reference), wherein the NAPs characterized to date have anticoagulant activity and / or serine protease activity. The present invention provides purified NAPs manufactured by the method of the claimed process, wherein said purified NAP is a NAP drug substance that can be formulated as a NAP drug product. The present invention may be particularly suitable for the manufacture of polypeptides that include at least one NAP domain. The present invention provides NAP drug substances and NAP drug products manufactured by the process described herein. In one embodiment, the present invention provides a process for the manufacture of an rNAPc2 / proline drug substance and an rNAPc2 / proline drug product, and provides the drug substance rNAPc2 / proline and the drug product r APc2 / proline manufactured by the process described here. Anticoagulant proteins extracted from Nematodes (NAPs), are so designated, because the first NAP originally isolated was extracted from a nematode, the canine uncinaria, Ancyclostoma caninum. The term "NAP domain" refers to a sequence that is considered to have anticoagulant properties. Generally, a NAP domain is an amino acid sequence that contains less than about 120 amino acid residues, and contains 10 cysteine residues, as described in U.S. Patent Nos. 5,863,894; 5,864,009; 5,866,542; 5,866,543; 5,872,098; and 5,945,275. The "NAP domain" can also refer to nucleic acid or nucleotide sequences that encode one or more amino acid sequences or polypeptides having NAP domains. Representative NAP domains, NAP amino acid sequences, broadly defined characteristics of this family of proteins and nucleic acid molecules, which encode said proteins, are described in U.S. Patent Nos. 5,863,894; 5,864,009; 5,866,542; 5,866,543; 5,872,098; and 5,945,275. The NAP drug substance of the present invention includes anticoagulants characterized by inhibiting blood coagulation, which includes plasma coagulation. The NAP drug substances of the present invention include, among others, those which increase the measured coagulation time of human plasma, in the prothrombin time (PT) and / or activated partial thromboplastin time (aPTT) assays, such and as described in U.S. Patent Nos. 5,863,894; 5,864,009; 5,866,542; 5,866,543; 5,872,098; and 5,945,275. One skilled in the art can use other assays to determine the anticoagulant activity of the NAP drug substances. One skilled in the art can also use other assays to determine other biological activities of the NAP drug substances. The terms "AcaNAPc2" or "rNAPc2" refer to recombinant proteins of the NAP family. The preparation and sequence of AcaNAPc2 is described in U.S. Patent No. 5,866,542. The terms "AcaNAPc2 / proline", "AcaNAPc2P", "rNAPc2 / proline" and "rNAPc2 / Pro" refer to a recombinant protein having the amino acid sequence of AcaNAPc2 which has been modified to add a proline residue in the C terminal of the AcaNAPc2 sequence. The terms "Drug Substance" or "Active Pharmaceutical Ingredient" (API) refer to pharmaceutically active material that can be further formulated with excipients to produce a drug product. The drug substance may be in bulk form. The term "Drug Product" refers to the finished dosage form (eg, capsules, tablets, liquid product in a bottle, lyophilized powder in a bottle) containing the substance of the drug in the final formulation buffer, and generally It contains inactive ingredients. The drug product may be a formulated drug substance. The term "Excipient" refers to the inactive ingredients intentionally added to the drug product, where it should be understood that the excipients do not have pharmacological properties in the amounts used. The term "Impurity" refers to a component present in a drug substance, API formulation, or drug product that is not the desired product, a substance related to the product, or excipient, wherein it is understood that an impurity may be present. related to the product or related to the process. The term "degradation products" refers to variants, especially to molecular variants, that result from changes in the drug substance or drug product over time due to light, pH, temperature, water or reaction with an excipient or a container / packing / wrapping system. The term "USP" refers to standards established in the Pharmacopeia of the United States of America (USP) and the National Form (NF) (United States Pharmacopeial Convention, Inc., Rockville Maryland (2002), "USP26-NF-21", whose total content is incorporated herein by reference), and the USP Reference Standards. Additional information can be found at http: //www.usp.org, or by consulting the USP-NF. The present invention provides a process that produces a high purity NAP drug substance, where raw material of animal origin was not used for any fermentation or purification step. This process can be scaled and is suitable to be operated on a product at a commercial production scale. The present invention provides a process for the manufacture of the drug substance NAP and the drug product NAP, wherein the process includes fermentation, recovery, purification, filtration and filling processes. A fermentation process is provided by means of which the NAP is produced in a suitable host, where the sequences encoding the NAP in the host genome are integrated. A recovery process is provided, which improves the yield and purity of the proteins recovered in the fermentation step, where the recovery process allows a more efficient capture of the NAP compared to the more conventional methodology, such as microfiltration and ultrafiltration. A purification process is provided whereby the substance of the NAP drug is purified from the contaminants, wherein the desired formulations are achieved using a combination of methods, including but not limited to, ultrafiltration, diafiltration, hydrophobic interaction chromatography, and ion exchange chromatography. An optional refill process is provided, wherein the NAP drug product is packaged and can be lyophilized. The processes of the present invention are suitable for the manufacture of NAP drug substances and NAP drug products. One skilled in the art can modify the processes described herein to improve the expression, recovery, purification, formulation or filling of a particular drug substance NAP. In a non-limiting example, one skilled in the art can determine the isoelectric point (pl) of a NAP of particular interest and can make minor adjustments to conditions, such as the binding capacity or pH of the chromatography step to achieve improved filtration of the desired NAP drug substance. The present invention provides purified NAP drug substances described herein from NAPs, including but not limited to AcaNAPc2, AcaNAPc2 / proline, AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4 AcaNAP24, AcaNAP25, AcaNAP46, and AcaNAP44. In particular, the present invention provides rNAPc2 / proline. One skilled in the art can identify other NAP proteins suitable for use in the processes described herein for the manufacture of purified NAP drug substances.

Fermentation The present invention provides a fermentation process in which NAP is produced in a suitable host. As proposed, one or more sequences coding for NAP are integrated into a host genome, and the host produces NAP during the fermentation process. In one embodiment, rNAPc2 / proline is produced as a protein secreted by Pichia pastoris in a fermentation process as provided herein. As provided herein, the fermentation process includes a seed fermentation process wherein the host cells are cultured at a desired cell density and a production fermentation process wherein the NAP is produced in a desired titer. A seed fermentation process provides a dense inoculum suitably for a fermentation process to produce high levels of NAP. The fermentation process proposed here also includes a production fermentation to produce high levels of NAP. The production fermentation includes different classes: a batch of glycerol; a batch fed glycerol; adaptation of methanol and induction of methanol. The glycerol batch phase builds the biomass. In the batch phase fed glycerol, a solution enriched with glycerol is fed to the culture to increase the biomass and suppress the expression. In the methanol adaptation phase, the glycerol feed is terminated and replaced by the methanol feed which induces the host to produce NAP. In the methanol induction phase, the processing conditions at the end of the methanol adaptation phase are maintained in order to maintain NAP production. According to one aspect, the pH range for fermentation is controlled to achieve the desired high level of NAP. In one embodiment, the pH for fermentation is controlled in a range of approximately 2.9 + 0.1 pH units during the methanol adaptation fermentation and the methanol induction fermentation. According to another aspect, the temperature for the fermentation is controlled. In one embodiment, the temperature of the methanol adaptation phase for fermentation is sustained at a temperature of approximately 28 ± 2 ° C for the first four hours to aid successful adaptation to the methanol feed and temperature for the remainder of the methanol adaptation phase, it is sustained at approximately 25 + 1 ° C to favor the high NAP titer. According to one aspect, the NAP title continues to increase without harmful effects on the product for seven days, which achieves a generally high level of NAP production.

In illustrative embodiments, the fermentation process provided herein has been carried out in fermenters of 15L, 100L, 150L and 1000L and the material of the fermentations of 15L, 100L and 150L has been purified to generate the drug substance NAP. In one embodiment, the fermentation process produces rNAPc2 / proline in a high titer. In several embodiments, the fermentation to produce rNAPc2 / proline has been performed in 15L, 100L, 150L and 1000L fermenters, and the rNAPc2 / proline drug substance has been purified from the 15L, 100L and 150L fermentations. Recovery The present invention provides a recovery process for improving the production and purity of the NAP proteins of the fermentation step. The recovery process provided here allows the capture of the NAP and the removal of cells and cell debris where the recovery process provided here is more efficient compared to the more conventional methodology, such as microfiltration / ultrafiltration. Without wishing to be limited by this theory, the improved efficiency of the recovery provided here may be the result of a combination of aspects of Pichla pastoris, that is to say that the Pichia pastorls creates a dense biomass during fermentation. In addition, NAP proteins are relatively small proteins and therefore, require small pore size filtration membranes, which have slow flow rates that result in long processing times. According to one aspect, the recovery process uses ion exchange chromatography, including the use of an expanded bed ion exchange chromatography unit to separate NAP from cells and cell debris, and to exchange the product in a regulator suitable for use in the following purification steps. In one embodiment, the Streamline SP XL ion exchange resin (Amersham Biosciences) of the expanded-bed chromatography unit is used to recover the substance of the NAP drug, as described herein. In another embodiment, the rNAPc2 / proline is separated from the cellular debris of the host cell by expressing rNAPc2 using expanded bed chromatography. In an especially preferred embodiment, the rNAPc2 / proline is recovered using the Streamline XL ion exchange unit. Alternatively, the recovery process for efficiently capturing the NAP from the fermentation step can be carried out using methods other than expanded bed ion exchange chromatography. An expert in the art can test and evaluate alternative methods for capturing NAP and removing cells and cellular debris, including but not limited to affinity chromatography, centrifugation, filtration, differential precipitation, and other methods that will be determined. Purification The present invention provides a purification process in which the substance of the NAP drug is purified from the contaminants. As provided herein, the purification process includes hydrophobic interaction chromatography, collecting the NAP fractions, at least one fraction of ultrafiltration and diafiltration (UF / DF) day NAP, ion exchange chromatography, the collection of NAP fractions of ion exchange chromatography and another UF / DF step, and a final filtration. It should be understood that each step provided in the purification process increases the purity of the substance of the NAP drug, so that one skilled in the art can determine the purity step required for a particular use and select the steps and conditions necessary to achieve the desired level of purity of the NAP drug substance. The overall process efficiency has been improved by maintaining a low pH (about 3) in the solution starting from the fermentation broth through the recovery step and the first purification step (Source 15PHE hydrophobic interaction chromatography).

These steps were specially designed to be carried out at the same pH, to eliminate the pH / regulator exchange steps required in other processes, thus reducing the time and manpower required, as well as reducing the potential for Product losses. These steps are carried out at a pH of less than about 5, preferably at a pH of less than about 4, more preferably at a pH of about 3. In one embodiment, hydrophobic interaction chromatography utilizes hydrophobic interaction chromatography media Source 15PHE at a pH of approximately 3.0 + 0.1. As provided herein, the purification process uses hydrophobic interaction chromatography to remove contaminants, where the use of a low pH allows the binding of large amounts of NAP to the hydrophobic media and the use of an elution gradient allows the separation of closely related impurities. As here provided, the fraction of NAP eluted from the hydrophobic interaction chromatography media passes through ultrafiltration and diafiltration (UF / DF) to concentrate the product and carry out the regulator exchange, after which, the NAP in a The appropriate regulator is applied to an ion exchange medium to remove most of the remaining protein and non-proteinaceous contaminants, including intimately related contaminants. Finally, as here provided, the fraction of NAP collected from ion exchange chromatography, which contains the substance of the highly purified NAP drug (API), goes through UF / DF to concentrate the substance of the NAP drug and exchange it in the formulation of final regulation. In one embodiment, the filtered conditioned eluate from the Streamllne SP XL chromatography step used for the recovery of NAP, is applied to a column of hydrophobic interaction chromatography media Source 15PHE (Amersham Biosciences), at a low pH (approximately 3.0 ± 0.1 ) where large amounts of NAP are bound to the column, followed by the Source 15PHE gradient elution, the addition of sodium hydroxide to raise the pH to about 5 or higher, and then the UF / DF of the NAP fraction eluted, after which the NAP solution is applied to a column of Source 15Q ion chromatography media (Amersham Biosciences) and the elution gradient is used to separate the drug substance NAP from the intimately related contaminants. In one embodiment, the NAP fractions of the ion exchange chromatography contain the substance of the NAP drug. In another embodiment, the purification process is carried out as described above to obtain highly purified rNAPc2 / proline drug substance.

As provided herein, solutions containing NAP can pass through several filtration steps to obtain the drug substance NAP at desired concentrations or in desired formulations. Additional filtration steps may be included as desired. Accordingly, the NAP fractions eluted from the chromatography steps can be filtered, concentrated, desalted, and go through the regulator exchange using ultrafiltration (UF) alone or in combination with diafiltration (DF) or a combination of ultrafiltration and diafiltration ( UF / DF). As provided herein, the UF / DF can be used to exchange the hydrophobic interaction chromatography elution buffer for the ion exchange chromatography charge regulator, or to exchange the ion chromatography elution buffer for a formulation buffer final, or a bulk drug formulation that is to be used for the NAP drug product. The UF / DF can be carried out using one or more filters. According to one aspect, a single filter (or filter membrane) is used, with a pore size of molecular weight selected for the UF / DF. Alternatively, multiple filters can be used, as necessary, for escalation. In one embodiment, the NAP fraction with the adjusted pH of the hydrophobic interaction chromatography step passes through ultrafiltration using a filter with a pore size of 3kDa MW to achieve the desired concentration, and then the retained pool containing NAP is diafiltered against 5 or more volumes of the exchange charge regulator, using the same filter membrane of pore size 3kDa MW until it is determined that the desired regulator conditions have been achieved. In one embodiment, the NAP fractions of the ion exchange chromatography pass at least one step to a final UF / DF. In another embodiment, the NAP fractions of the ion exchange chromatography step pass through the UF / DF as described above to exchange the substance of the NAP drug in the regulator of the final drug formulation. In another embodiment, regenerated cellulose ultrafiltration filters with a pore size of 3kDa MW are used for ultrafiltration or diafiltration. One skilled in the art can select and evaluate filters and filter membranes that are suitable for, and compatible with, the experimental conditions and the desired goals. Bulk Filtration As here provided, the substance of the drug NAP in final formulation regulator can be filtered and stored, and go through additional processing steps. In one embodiment, the substance of the NAP drug in the final formulation goes through a filling process. In one embodiment, the substance of the bulk NAP drug is transferred to a suitable sterile environment, for example, "class 100 area" in a manufacturing facility, and filtered in sterile containers. In another embodiment, the substance of the NAP drug is filtered, for example, using a 0.2 μ filter, and the containers of the suitable material are autoclaved, ie, containers made of fluorinated ethylene propylene (FEP), copolymer of ethylene tetrafluoroethylene (EFTE), or other material that meets the requirements of the Food Additives Amendment of the Federal Drug, Food and Cosmetic of the United States of America, and the Class VI designation of the USP. In one embodiment, the product of the bulk drug rNAPc2 / proline is transferred to a Class 100 area and filtered using a 0.2 μ filter? Millipak in a 1 liter bottle of Tefzel® FEP 1600 molded Nalgen, uncoated, and a non-contaminated Tefzel®ETFE screw closure after which the bottles are transferred to a freezer at a temperature between -20 ± 10 ° C for storage. The substance of the bulk NAP drug can be filtered again and filled using the same method for the final filtration, that is, the content of smaller bottles filled as described above, can be transferred to a larger container. In one embodiment, the contents of the FEP Teflon bottles containing the rNAPc2 / proline drug substance as described above are transferred in a carboy Class 100 area autoclaved, refiltered and filled. Faso of Filling According to one aspect, the present invention further provides a filling step, wherein the substance of the NAP drug in the final formulation of the drug is filled into a bottle, container or other aseptic package. The filling step may include additional filtration steps, and may include the use of the previous filling suite to fill individual jars, containers and other packages. A filling step can provide a vial of the drug product NAP, wherein the drug product NAP is in the final dosage form. A liquid fill step can provide a vial of the NAP drug product in liquid form. The product of the NAP drug can be used in the form introduced during the filling step, i.e., a unit dose of solution containing the NAP drug product. Alternatively, the formulation of the NAP drug product can be further manipulated after the filling step, ie, the NAP drug product from a bottle after a liquid filling step can then be lyophilized. As provided herein, the substance of the NAP drug in the desired final concentration can be filtered in an aseptic filling suite and then filled into previously sterilized individual vials. In one embodiment, the substance of the rNAPc2 / proline bulk drug, (in a concentration of 12 ± 1.2 mg / mL) is diluted in 3 mg / mL in a solution of 0.2 alanine, and 25 mM of monobasic sodium phosphate, pH 7.0 The rNAPc2 / diluted proline is then diafiltered with > 5 volumes of an alanine / phosphate solution. The rNAPc2 / proline solution is removed and the filters are rinsed with alanine / phosphate solution. The diafiltered rNAPc2 / proline solution is then diluted to 2 mg / mL (as measured by the UV test described below in the examples) with the filter rinses, and the alanine / phosphate solution. The 2 mg / mL solution of rNAPc2 / proline is then diluted with an equal volume of 25 mM sodium phosphate, 8% sucrose, pH 7.0 to achieve a concentration of 1.0 ± 0.1 mg / mL rNAPc2. Finally, the formulated rNAPc2 / proline solution of 1 mg / mL is filtered using a 0.2 μ? t filter? Millipak (Millipore Corp.) before the filling step. The rNAPc2 / proline drug product in 1 mg / mL is filtered in an aseptic filling suite through two 0.2 filters. Millipak online. The rNAPc2 / proline is then filled into 3-cc glass jars previously sterilized and partially capped. As an alternative embodiment, the substance of the bulk rNAPc2 / proline drug can be formulated for a liquid drug product. This is described in example 5.1. Lyophilization The present invention provides an optional lyophilization step to produce the lyophilized NAP drug product. After the filling step, the NAP drug product in the bottles or other containers are freeze dried and then sealed, that is, the bottle caps are well fitted and the bottles are capped. The lyophilized formulation maintains high purity and sustained stability when the NAP drug product is subjected to a severe temperature stress, for example, 28 days at a temperature of 50 ° C. The present invention will be further explained by means of specific examples that are presented below for the purpose of showing the characteristics of the processes for the manufacture of rNAPc2 / proline and the characteristics of the r APc2 / proline produced by these processes, including data and methods of characterizing the purified product. In the following examples, the aforementioned effects are clarified by describing the processes for the manufacture of the rNAPc2 / proline drug substance suitable for the formulation as drug products for use in a pharmaceutical composition. However, these embodiments are set forth to illustrate the invention and should not be construed as limiting thereof, the present invention being defined by the claims. EXAMPLES Example 1. Preparation of cell banks for the drug substance expression system NAP Example 1.1. Expression System of rNAPc2 / proline The rNAPc2 gene was cloned into a Pichia pastoris expression vector, pYAM7sp8 (Laroche et al., 1994, Biotechnology 12: pages 1119 to 1124), using the PCR rescue. The vector pYAM7sp8 (figure 1) is a derivative of pHIL-D2 (Despreaux and Manning, 1993, Gene 106: pages 35 to 41). This vector contains the promoter and the transcription termination signal of the AOX1 gene of Pichia pastoris, a secretion signal peptide (a fusion of the acid phosphatase signal sequence of Pichia pastoris and the pro-sequence of a factor of hybrid a-coupling of S. cerevisiae), and the HIS4 marker to select the transfectors. The PCR primers used to rescue the rNAPc2 / proline gene from the phage clone (Jespers et al., 1995, Biotechnology 13: pages 387 to 382) were: A8: 5GCG TTT AAA GCA ACG ATG CAG TGT GGT G 3 '( SEQ ID NO: 1) A9: 5 C GCT CTA GAA GCT TCA TGG GTT TCG AGT TCC GGG ATA TAT AAA GTC 3 '(SEQ ID NO: 2) These primers add the sites Dra1 and Xba \ to the 5' and 3 'ends, respectively, of the rescued fragment of DNA. The underline indicates the nucleotides that hybridize to the template. Primer A9 (SEQ ID NO: 2) also inserts a proline codon just before the stop codon, which converts the coding sequence from an AcaNAPc2 coding (SEQ ID NO: 3) to an AcaNAPc2 / proline coding (SEQ ID NO: 4) The resulting PCR fragment was digested with Dra1 and Xoa1 and cloned into pYAM7sp8 digested with Si1-1 and Spe1. Ligand blunt ends of pYA 7sp8 (Sfu1) and the PCR fragment (Dra1) resulted in a fusion within the P secretory signal peptide of P. pastoris for the mature portion of the rNAPc2 / proline. Linking the Xba1 and Spe1 ends of the PCR fragment and pYAM7sp8 resulted in the destruction of the pYAM7ps8 Spe1 site. The P. pastoris expression strain was constructed by integrating the expression cassette into a genome of P. pastoris through homologous recombination. The construction pYAM7sp8 / NAPc2 was digested with Aor1. The digested plasmid was electroporated in P. pastoris GS115 cells (his4-). The transfectors were selected for methanol utilization phenotype (mut +) and high level expression of rNAPc2. A single isolate (designated as GS 115 / AcaNAPc2P-55) was selected for the generation of the Master Cell Bank (MCB). The production strain was analyzed by Southern blots that were tested by radiolabeled rNAPc2 or HIS4 genes. These examinations showed that multiple copies of the expression cassette were integrated into the 3 'site of the AOX1 gene. Example 1.2. Master Cell Bank (MCB) The Master Cell Bank (MCB) was prepared using a previous bank of a single colony isolate (GS115 / AcaNAPc2P-55). The flask containing the medium of the YEPD bottle (bacto peptone, yeast extract and dextrose) with 2% glucose was incubated with 0.5 mL of the pre-bank and cultured at an optical density (A550nm) of 0.5 to 1.0. The culture was harvested, diluted with glycerol to a final concentration of 15% as a cryo-preservative, and frozen in cryo-flasks stored at a temperature below -60 ° C. Example 1.3. Manufacturer's Working Cell Bank (MWCB) A new Manufacturer's Working Cell Bank (MWCB) was manufactured from a bottle of MCB. The MCB bottle was used to inoculate a vial containing yeast Peptone medium (peptone and yeast extract) and 2% dextrose. The bottle was incubated at a temperature of 28 ± 2 ° C and 250 rpm until the optical density (A6oonm) was 17.0 ± 5.0. The culture was harvested, and glycerol was added as a cryo-preservative, at a final concentration of 9%. Aliquots of 1.1 ± 0.1 mL were filled into 2.0 mL cryo-flasks, which were frozen and stored at a temperature of -70 ± 10 ° C. Example 1.4. Test Methods Used for the Analysis of Master Cell Bank Identification of the Guest. The culture of the rNAPc2 / proline cell bank was streaked on Trypticase Soy Agar plates (TSA), and the plates were incubated for culture. The isolate was prepared for identification using a Vitek® identification system which uses a temperature controlled chamber and a photometric sensor unit to monitor changes in the turbidity of the isolated suspension, which had been inoculated into the test card. Vitek® yeast containing substrates for 26 conventional biochemical tests. For the host identifications of the rNAPc2 / proline cell bank, the resulting bio-reaction pattern was compared to a positive control organism (Pichia pastoris, ATCC No. 76273) bio-reaction pattern. Viable Cell Concentration The concentration of viable cells of the rNAPc2 / proline cell bank was measured by enumerating the viable colony forming units (CFU) by preparing serial dilutions of three flasks from the cell bank (each from the start)., the middle part and the end). Dilutions were plated in triplicate plates on TSA plates and incubated, CFUs are counted and calculations are performed to determine cell concentration as CFU / mL. Structural Sequence Analysis of the Gene. The cell bank culture was prepared for the sequence formation of the gene by amplifying the rNAPc2 / proline gene incorporated in the host genome using the Polymerase Chain Reaction (PCR) technique. The PCR product was purified and the concentration determined. A sequence of the PCR product was then elaborated, using dideoxy chain termination methods (Sanger). The sequence of the resulting gene from the cell bank was compared to a known DNA sequence of rNAPc2. The identity was confirmed by a 100% comparison. Contamination Test that is not from the Guest. The fermentation broth of rNAPc2 / proline was tested for contamination for non-hosts by inoculating 100 ml_ in each of ten TSA plates. Three plates were incubated at three temperatures (20-25 ° C, 30-34 ° C and 35-39 ° C). During the seventh day of the incubation period, the plates were inspected to determine the microbial colonies that differ from the characteristic host, noting particularly the differences in the colony morphology, color and / or size of the colony. The Gram strain was also made on the final reading plate. The appropriate negative controls were incubated in the assay. Example 1.5. Test Methods Used for the Analysis of the Bank of Work Cells of the Manufacturer Identification of the Guest. The culture of the rNAPc2 / proline cell bank was streaked on Sabouraud Dextrose Agar plates (SDA) and the plates were incubated for culture at a temperature between 20 ° C and 25 ° C for 7 days. In parallel, a positive control (ATCC strain of K. pastoris, an alternate name of P. pastoris) was scored on SDA plates in the same manner. The selected colonies that grew then were tested by gram staining. After incubation, at least two morphologically similar colonies from each SDA plate were selected for the SDA plates from the test sample and the SDA plate from the positive control. These colonies were subcultured on separate SDA plates and incubated at a temperature of 20 ° C to 25 ° C for 7 days. The API 20C AUX test and the Gram stain were then performed on the proliferation of each sub-culture plate. The API test system (bioMérieux SA; Marcy l'Etoile, France) is a manual microbial identification test that contains 20 miniature biochemical tests. The 20C AUX test strip contains 20 specific biochemical tests for the identification of yeast. The results of the API test for the test sample of the rNAPc2 / proline cell bank were compared with the results obtained for the positive control to confirm the identification. Viable Cell Concentration The concentration of viable cells of the rNAPc2 / proline cell bank was measured by enumeration of viable colony forming units (CFUs) by preparing serial dilutions of two cell bank flasks, extracted one flask prior to bank freezing. and a bottle after the freezing of the bank. An aliquot of 100 μ? of each dilution was placed in TSA plates in duplicate and were incubated for a period of 5 to 7 days. All plates with counting colonies (30-300 CFUs) were counted. The counts obtained from the plates of the same dilution of the test sample were averaged, multiplied by that dilution, and divided by the aliquot size of 100 μm to report the results as CFU / mL.

Preparation of DNA Sequence. The total DNA was isolated from the newly created cell bank (test article). The NAPc2 / proline gene was amplified by polymerase chain reaction (PCR) using primer homologs for the 5 'and 3' sequences of the cloned NAPc2 / proline gene. The resulting DNA fragment (approximately 500 bp) was purified using standard methods and used as a template for the DNA sequence elaboration using a primer pathway strategy using a Termo labeled radio cycle terminator sequence preparation kit. Sequenase (Amersham Biosciences, Piscataway, NJ). Sequence processing films were read by digitization and sequence data assembled and analyzed using Sequencher ™ software, version 3.0 (Gene Codes Corp., Ann Arbor, MI). The consensus sequence produced from the test article was then compared to the theoretical sequence for the NAPc2 / proline gene. Contamination that is not from the Guest. Prior to the freezing of the newly created cell bank (test article), a vial for the non-host test was submitted. A sample of the broth was diluted one hundred times in saline. Duplicate plates of nine different media types were inoculated with 100 μl of the diluted test sample. In addition, a positive control (ATCC strain of K. pastoris, an alternate name of P. pastoris) was diluted and inoculated on plates in the same manner. Another set of plates was not inoculated and was designated as the plates of the negative controls. All plates except the SDA plate were incubated at a temperature of 30 ° C to 35 ° C for a period of 48 to 72 hours; SDA plates were incubated at a temperature of 20 ° C to 25 ° C for 7 days. The plates were examined for growths after days 1 and 2 or 3. In addition, the SDA plates were examined for growth after 7 days. Any aberrant colonies were identified by the API test and Gram stain. After day 2 or 3, at least two morphologically similar colonies of each TSA plate were selected from the test sample TSA plates and the positive control TSA plates. These colonies were subcultured on separate TSA plates and incubated at a temperature of 30 ° C to 35 ° C for a period of 48 to 72 hours. The API 20C AUX test and gram staining were then performed on the proliferation of each sub-culture plate. The API test system (bioMérieux SA, Marcy l'Etoile, France) was a manual microbial identification test containing 20 miniature biochemical tests. The 20C AUX test strip contains 20 specific biochemical tests for the identification of yeast. The results of the API test for the test article were compared with the results obtained by the positive control to confirm the identification. Example 2. Manufacture of the Drug Substance of rNAPc2 / proline The manufacturing process for the production of the rNAPc2 / proline drug substance consists of fermentation, recovery, purification, bulk filtration and filling. The following sections describe the operations of the individual units of each stage of the process. The flow charts for each operation of the unit are presented in figures 2 through 4, where the diagrams summarize the equipment, regulators, components and input and output parameters. The substitution of suppliers and materials can be covered, as necessary, while maintaining compliance with the requirements of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Active Pharmaceutical Ingredient (API) Good Manufacturing Protocol ( GP) requirements. Example 2.1 Fermentation This section describes the fermentation process for the production of rNAPc2 / proline. The rNAPc2 / proline protein was produced as a protein secreted by Pichia pastoris. The fermentation process for rNAPc2 / proline consisted of seed jars, a fermentation of seed and a production fermentation (Figure 2, Fermentation Flow Diagram). All the components of the media used Purified Water, USP. Seed Bottles for Seed Fermentation. The purpose of the unit unit seed jar unit operation was to provide a dense inoculum suitable for seed fermentation. Three bottles of MWCB were thawed and one milliliter was used to aseptically inoculate each of the three diverted agitation flasks of two liters for a content of 250 mL of medium autoclaved at a pH of 6.0 ± 0.1 (Table 1 ). The flasks were covered and transferred to the shaker of the coater at a speed of 250 ± 5 rpm and a temperature of 2 ° C. The flasks were covered for a period of 27.5 + 2.0 hours, until the cell density measured by the weight of wet cells (WCW) was >; 30 g / L. Once these two parameters were achieved, the content of the two bottles was transferred aseptically to an inoculum bottle subjected to autoclaving.

Table I. Seed Bottle Medium Seed Fermentation The purpose of the fermentation of the seed was to provide an adequately dense inoculum for the production fermentation. The medium for seed fermentation (table 2) including PTM4 Trace Salts (table 3) was transferred to a seed warmer. The medium was sterilized by steam, allowed to cool and the pH was adjusted to 5.0 ± 0.2 with ammonium hydroxide from 28% to 30% sterilized by filter. An antifoam solution sterilized by 5% (v / v) filter KF088 in 50% methanol was then added through a stopper in a concentration of 0.5 M 1/1. When the temperature was stabilized at a temperature of 28.0 + 1.0 ° C, the medium was inoculated with the content of the inoculum bottle of the seed bottle in a proportion of 2.5%. the pH of the fermentation culture was maintained at a pH of 5.0 ± 0.2 with ammonium hydroxide of 28% to 30%. The proliferation of the fermentation was monitored by measuring the weight of the wet cells (WCW). The fermentation was carried out for a period of 15 ± 5 hours up to a final wet cell weight of > 20 g / L. A portion of the seed fermentation culture was transferred through a sterilized transfer line by steam to an inoculum canister autoclaved. A sample of final seed fermentation was tested to determine non-host contamination.

Table 2. Seed Fermentation Media Table 3. Trace salts PTM4 Components Concentration Cupric Sulfate, Pentahydrate 2.0 g / L Sodium Oodide 0.08 g / L Sodium Molybdate, Dihydrate 0.2 g / L Zinc Chloride 7.0 g / L Ferrous Sulfate , Heptahydrate 22.0 g / L Boric Acid 0.02 g / L Cobalt Chloride, Hexahydrate 0.5 g / L Manganese Sulfate, Hydrohydrate 3.0 g / L d-Biotin 0.2 g / L Sulfuric Acid 1 , 0 mUL Production Fermentation The purpose of the production fermentation was to produce high levels of rNAPc2 / proline protein. To achieve this, the culture was cultured in a high density of cells before induction of the rNAPc2 / proline gene. The medium for the production fermentation (table 4) was prepared in a production fermentor. These media components were dissolved and mixed with purified USP water and then steam sterilized. The tank was cooled to its initial operating temperature of 28.0 ± 1.0 ° C. then an antifoam solution sterilized by 5% (v / v) KF880 filter in 50% methanol was added. The pH was adjusted to its initial operating range of 5.0 ± 0.3 with ammonium hydroxide sterilized by filter from 28% to 30%. When the initial conditions of operation were achieved, the medium was inoculated with the content of the inoculum of fermentation of seed of the can in a proportion of 1 kg of inoculum per 10 kg of the medium stored initially (pre-inoculation weight).

Table 4. Production Fermentation Medium The fermentation of the medium consisted of four different phases: glycerol batch, feed glycerol batch, methanol adaptation and methanol induction. Throughout the fermentation, the dissolved oxygen levels were maintained at approximately 35% by the addition of air at a constant rate and the use of variable back pressure and agitation. If additional oxygen was needed once agitation had been achieved, the water jet was supplemented with oxygen gas. The pH of the culture in the fermenter was maintained with ammonium hydroxide of 28% to 30%. The antifoaming solution was added periodically to control foam formation. The first phase of fermentation, the phase of the batch of glycerol, construction of biomass. The fermentor was operated at a temperature of 28 ± 2 ° C until the glycerol in the medium was depleted, as detected by an oxygen peak caused by the cessation of the metabolism of giicerol. This was followed by the fed batch phase of giicerol in which a 50% w / w solution of giicerol was fed to a culture in an amount of 18.0 + 1.0 mL / kg pre-inoculation / hour for a total of 8.5 hours to increase the biomass and repress the expression. During the first 4.5 hours of this phase of giicerol feeding, the pH adjustment point of the culture was reduced from 5.0 ± 0.3 to 2.9 ± 0.1 at an index of 0.5 pH units per hour and kept at this pH the rest of the fermentation, that is, for the duration of the induction of the gene induced by methanol. The temperature was maintained at 28 ± 2 ° C during this entire phase. The WCW was from >; 225 g / L before the end of the phase of the giicerol feed batch. In the methanol adaptation phase, the giicerol feed was terminated and replaced by the methanol feed, which induces the organism to produce rNAPc2. The methanol feed (with a content of 6.0 mL / FO880 antifoam) was started at a weight prior to inoculation of 3.0 mL / kg / hour. The culture was tested for the methanol adaptation starting at 2 hours after the initiation of the methanol addition. The methanol adaptation test consisted of briefly determining the feed and verifying a peak in the dissolved oxygen. After the first four hours of the methanol addition the temperature was lowered to a temperature of 25 + 1 ° C for a period of 2 hours. After the first four hours of methanol addition and after verification that the crop was using methanol, the methanol feed rate was increased by 1.0 mL / kg prior to inoculation / hour. The methanol consumption was measured every hour to ensure that methanol was being completely exhausted, at which point the methanol feed rate was increased by 1.0 mL / kg of weight prior to inoculation / hour, up to a final feed rate of 6.0 mL / kg of weight prior to inoculation / hour. During the methanol induction phase, the final processing conditions of the methanol adaptation phase were maintained throughout the remaining implementation. Starting at approximately 48 hours of total fermentation time, the production of rNAPc2 / proline was monitored by determining the concentration of the broth supernatant measured by a C8 Reverse Phase assay. The production fermentor was harvested after 144 to 168 hours in the production fermentor, and after the concentration of rNAPc2 / proline measured by the Inverse Phase C8 test was = 0.55 g / L. A sample of the final fermentation was tested to determine non-host contamination.

The pH was maintained at a pH of 2.9 ± 0.1 during the adaptation phase of methane! and the methanol induction phase. The fermentation broth had a pH of 2.9 ± 0.1. Example 2.2 Recovery This section describes the recovery procedures for the production of rNAPc2 / proline. The recovery process for the rNAPc2 / proline consisted of an expanded bed chromatography unit operation as shown in the flow chart of Figure 3. Streamline Ion Exchange Chromatography The purpose of the Streamline SP ion exchange chromatography step XL was to separate the rNAPc2 / proline from cell debris and exchange the product in a suitable buffer for a first step of purification chromatography. The medium used to achieve the separation was the column of expanded-bed ion exchange chromatography of Streamline SP XL resin (Amersham Biosciences). The fermentation broth (at a pH of 2.9 ± 0.1) was diluted with purified water until the conductivity was > 9 mS / cm. The solution was adjusted to a concentration of 150 mM acetate and the pH was adjusted to a pH of 3.1 ± 0.2 using 17.4 M acetic acid. The loading solution was applied to the expanded resin bed which had been equilibrated with 500 mM of acetate sodium, pH 3.2 followed by 50 mM sodium acetate, pH 3.2. The column was washed in the upflow mode with 50 mM sodium acetate, pH 3.2 and then 50 mM sodium acetate / 150 mM NaCl, pH 3.2. The resin bed was allowed to settle and an additional wash was performed using 50 mM sodium acetate / 150 mM NaCl, in a downflow mode. The rNAPc2 / proline was eluted by the application of 50 mM sodium acetate / 350 mM NaCl, pH 3.2, and the concentration of rNAPc2 / proline was measured by a C8 Reverse Phase assay. In the preparation of the 15PHE purification chromatography step, solid sodium sulfate was added to the Streamline eluate at a final concentration of 0.85M. The pH was adjusted to a pH of 3.1 ± 0.2 using 2.4 M citric acid, and the conductivity was verified to be 100 ± 10 mS / cm. The conditioned Streamline eluate was filtered through 0.45 pm filters. Example 2.3. Purification This section describes the purification procedures for the production of rNAPc2 / proline. The purification manufacturing process for rNAPc2 / proline consisted of a hydrophobic interaction chromatography step, an ultrafiltration and diafiltration step, an ion exchange chromatography step followed by an ultrafiltration / diafiltration step and a final filtration and filling of the drug substance rNAOc2 / proline, also called the Pharmaceutically Active Ingredient (API), as shown in Figure 4. Source 15PHE Hydrophobic Interaction Chromatography The initial purification step partially purified the product by removing some contaminants from the protein and non-proteinaceous of rNAPc2 / proline using hydrophobic interaction chromatography column Source 15PHE (Amersham Biosciences). The filtered, conditioned Streamline eluate was applied to the Source 15PHE column previously equilibrated with 50mM sodium citrate / sodium sulfate 1.1M, pH 3.0. After loading, the column was washed with equilibrium regulator. The rNAPc2 / proline protein was eluted from the column using a volume column gradient of 1.1 M to 0.3 M sodium sulfate in 50 mM sodium citrate, pH 3.0, followed by a steady gradient of 0.3 M sodium sulfate until that the UV absorbance returned to the baseline. Fractions were collected at the elution peak rNAPc2 / proline and then analyzed by the Inverse Phase C18 assay. Fractions containing high purity purity of rNAPc2 / proline were pooled and tested by concentration by the UV test. The pH of the Source 15PHE set was adjusted to a pH of 5.3 ± 0.1 by the addition of 5N NaOH.

Step # 1 Ultrafiltration / Diafiltration (UF / DF "1) The purpose of UF / DF # 1 was to concentrate the product and exchange the rNAPc2 / proline in the regulator used for the 15Q chromatography, ultrafiltration filters of regenerated cellulose were used. of a molecular weight pore size of 3kD The Source 15PHE set with the adjusted pH was concentrated at 2.0 + 0.5 g / L (measured by UV Concentration) in the UF / DF # 1 membranes that had previously been equilibrated with 50m of sodium acetate, pH 5.3, the whole was then diafiltered with> 5 volumes of 50mM sodium acetate, pH 5.3, and until the pH was 5.3 ± 0.1 and the conductivity was <6.0 mS / cm. UF / DF # 1 diafiltration set was filtered through a 0.2 μ filter in preparation for loading on the Source 15Q column Source 15Q Ion Exchange Chromatography The operation of the final chromatography unit eliminated most of the the remaining protein and non-proteinaceous contaminants of the rNAPc2 / proline using a column of ion exchange chromatography media Source 15Q (Amersham Biosciences). The filtered UF / DF # 1 set was applied to a column of Source 15Q chromatography previously equilibrated with 500mM sodium acetate, pH 5.3 followed by 50mM sodium acetate, pH 5.3. After loading, the column was washed with 50 mM sodium acetate, pH 5.3 in equilibrium buffer. A linear volume gradient of column 20 from 0 to 400 mM NaCl in 50 mM sodium acetate, pH 5.3 was applied to the column. The fractions were collected at the elution peak and analyzed by the C18 Reverse Phase test. Fractions containing high purity purity of rNAPc2 / proline were pooled and tested by the UV test concentration. Final Ultrafiltration / Diafiltration Step (Final UF / DF) The purpose of the Final UF / DF was to concentrate the product and exchange the rNAPc2 / proline in the final formulation buffer. Ultrafiltration filters of regenerated cellulose with a molecular weight pore size of 3kD were used. The Source 15Q set was concentrated at 12.0 ± 0.5 g / L (measured by the UV concentration), in the UF / DF Final membranes, previously equilibrated with the final formulation regulator, 65mM sodium phosphate / 80mM sodium chloride, pH 7.0. The set was then diafiltered with > 6 volumes of the formulation regulator, until the pH was 7.0 ± 0.1. Example 2.4 Bulk Filtration and Filling The purified rNAPc2 / proline API was transferred in a Class 100 area and filtered using a 0.2 μm Millipak filter into a 1 liter bottle of Tefzel® FEP 1600 molded Nalgen (fluorinated ethylene propylene) ), with a non-polluting screw closure without molded coating Tefzel® ETFE (ethylene tetrafluoroethylene copolymer). The bottles were transferred to a freezer at a temperature of -20 ± 10 ° C for storage. The bulk API can be filtered again and filled using the same method that was used for the final filtration. The contents of the Teflon FEP bottles were transferred to the carboy subjected to the autoclave in the Class 100 area, filtered again and filled. With respect to storage containers and closures: both the FEP and the ETFE cover the requirements of the Food Additives Amendment of the Federal Food, Drug and Cosmetic Act of the United States of America. The material met the requirements of the USP Class VI designation. Example 3. Controls in the Process: rNAPc2 / proline Test Methods in Process The conditions that were monitored, including the acceptance criteria in the process, are found in the list of process flow diagrams (figures 2 a the 4). Below are brief descriptions of the test methods in process. RNAPc2 Test Methods in pH Process. It was read a sample using a pH meter that had been calibrated with pH standards that can be tracked NIST immediately before the test. The pH of the sample was read at a temperature of 25 ± 2 ° C. Conductivity. The electrolytic components of the solution were measured using a conductivity meter that had been standardized with the supports of the range conductivity standards that were to be measured. The conductivity of the sample was read at ~ 25 ° C. Weight of Humid Cells. Approximately 1.5 μL of fermentation samples were added to calibrated microcentrifuge tubes and centrifuged at 10 ° C., 000 rpm for approximately 5 minutes. The supernatant of each tube was decanted and the tubes containing the solids were weighed. The weight of the wet cells was equal to the net weight divided by the volume of the original sample. Contamination Test that is not from the Guest. The final broth from the seed and production fermentation samples was tested for final contamination of the host by inoculating 100 μL on each of the nine TSA plates. Three plates were incubated at three temperatures (20-25 ° C, 30-34 ° C and 35-39 ° C). During the seven-day incubation period, the plates were checked for microbial colonies that differed from the characteristic host, observing particularly the differences in the colony morphology, color and / or size of the colony. Gram stain was also performed on the final reading date. Appropriate negative controls were included in the trial. Reverse Phase C8 Test (Concentration and Purity).

The supernatants from the production fermentation samples and the Streamiine SP XL samples were filtered on 0.22 μ? T filters. and then injected into a Kromasil C8 Reverse Phase column, 4.6 x 250 mm. The column was equilibrated with 22% acetonitrile, 0.1% trifluoroacetic acid (TFA) before injection of the sample. A linear gradient of 22% to 28% acetonitrile in 0.1% TFA was then operated for twenty minutes in a range of 1 mL / minute to elute the rNAPc2 / proline material. Standard dilutions in rNAPc2 / proline that had known concentrations were used to generate a standard curve based on the linear regression of rNAPc2 / proline mg / mL against the peak area. The amount of rNAPc2 / proline in any sample was extrapolated from the standard curve and divided by the volume of injected samples to determine the concentration of rNAPc2 / proline in the samples. The purity of rN APc2 / proline was calculated as a percentage of the total peak area. Concentration by UV. The concentration of each purification set of the 15PHE chromatography through the final UF / DF step was determined using the absorbance at 280 nm in a properly calibrated spectrophotometer. The instrument was zeroed using the applicable regulator and in solution before the test samples were operated. The test samples were prepared by diluting within a linear range (between 0.13 -1.62 AU). The mean absorbance at 280 nm was divided by the extinction coefficient [0.59 AU / cm "1 (mg / mL) '1] and multiplied by the dilution factor to obtain the concentration in mg / mL Reverse Phase C18 Assay ( Purity) The purity of the Source 15PHE fractions and the set, the UF / DF # 1 set, the Source 15Q fractions and the set, and the final UFDF set were each analyzed by a Reverse Phase C18 assay. proline was separated from other components of a sample by linear gradient reverse phase chromatography.The samples were diluted, if necessary, to approximately 1 mg / mL in cPBS and 30 pL was injected into the C18 Waters Symmetry Reversed Phase column ( particles of 5 μm, length 4.6 mm ID x 250 mm, Waters Corp., Bedford MA) equilibrated in 78% of mobile phase A (0.1% of TFA in water), and 22% of mobile phase B (0.1% of TFA in acetonitrile) The percentage of mobile phase B was then linearly increased to 26% during a Twenty minute time period, using a flow rate of 1 mL / min. The peaks were monitored by the UV detector at 210 mm. The purity of rN APc2 / proline was calculated by dividing the peak area of rNAPc2 / proline by the area of the total peak in the chromatogram and expressing that proportion as a percentage. Example 4. API Test Methods of rNAPc2 / proline Appearance, Ph, and concentration: an aliquot of the test article was examined visually for color, clarity and the presence of any visible foreign material. A sample was read using a pH meter calibrated with pH standard that can trace NIST immediately before the test. The pH of the sample was read at 25 ± 2 ° C. The concentration of the sample was determined using its absorbance at 280 nm in a properly calibrated spectrophotometer. The instrument is bleached using a sample diluent before operating the test samples. Test samples are prepared in triplicate by diluting them within a linear range of (0.13 -1.62 AU) established during the validation of the method. The average absorbance at 280 nm is divided by 0.59 AU / cm-1 (mg / mL) "1 (the extinction coefficient) and multiplied by the dilution factor to obtain the concentration in mg / mL. rNAPc2 / proline test standard and rNAPc2 / proline reference standard were reduced and alkylated before enzymatic digestion.RNAPc2 / proline was denatured by treatment with a high concentration of guanidine hydrochloride, and then reduced with dithiothreitol. The reduced cysteines were then alkylated with iodoacetamides. Reduced and alkylated rNAPc2 / proline preparations were digested with 2% w / w trypsin for approximately 16 hours at a temperature of 37 ± 2 ° C. The tryptic peptides from each of the digested rNAPc2 / proline protein samples were then separated by reverse phase chromatography to generate a fragment pattern in the form of a chromatogram or "fingerprint". The elution profile of the sample was compared visually with a standard elution profile using peak retention times. The profiles must be compared, without new or eliminated peaks. Coomassie SDS-PAGE (Identity / Purity): The test samples, reference standard rNAPc2 / proline and the intensity marker of rNAPc2 / proline were diluted, with or without a reducing agent, using a Novex LDS Sample Preparation Regulator NuPAGE® (pH 8.4) at a final concentration of 0.5, 0.5, and 0.005 mg / mL, respectively. A mixture of protein standards (Novex Mark 12®) was diluted according to the manufacturer's instructions. The reduced samples were heated for five minutes at a temperature of 95 + 2 ° C. The reduced and unreduced samples were operated in separate gels. The sample loads of ten μg of the reference standard and the test sample, a sample load of 0.1 pg of the intensity marker (1% sample charge), and the appropriate mass of the ark 12 standard were analyzed by electrophoresis. in Novex NuPAGE® before casting from 4 to 12% of acrylamide Bis-Tris gel at a pH of 6.4. The gels were stained with a coomassie colloidal blue Novex dye. To confirm the identity, the main band was compared visually with the reference standard and with the mixture of the protein standards. The intensity of any of the impurity bands was visually prepared with the 1% intensity marker band. Any band of impurity greater than the marker was reported. If no impurity band greater than the marker was present, the purity was reported as being comparable to the reference. Note that the rNAPc2 / proline band did not appear in the desired molecular size. Due to its non-spherical shape, rNAPc2 / proline runs at a larger apparent molecular size. The rNAPc2 / proline band ran between 21.5 and 31 kDa standards in non-reduced gels while the rNAPc2 / proline band was operated at a standard of 21.5 kDa in reduced gels (Novex are products of Invitrogen Corp., Carlsbad CA). Reverse Phase C18: rNAPc2 / proline was separated from the other components of a mixture by linear gradient reverse phase HPLC. The purity of the rNAPc2 / proline was reported as the ratio of the peak area of rNAPc2 / proline divided by the total area of the peak in the chromatogram, expressed as a percentage. Injected into the C18 Waters Symmetry Reverse Phase column volumes of thirty μl of dilutions of the sample at approximately 1 mg / mL in cPBS (5 μm p particles, length 4.6 mm ID x 250 mm, Bedford A) balanced in 78% of mobile phase A (0.1% of TFA in water) and 22% of mobile phase B (0.1% of RFA in acetonitrile). The percentage of mobile phase B was then linearly increased to 26% over a period of twenty minutes, using a flow rate of 1 mL / min. The peaks were monitored by the UV detector at 210 nm. Endotoxin: Endotoxin measurements were made according to the UPS Bioactivity method: the rNAPc2 / proline extended the clotting time of human plasma initiated by the addition of thromboplastin in a concentration-dependent manner. The anticoagulant effect of rNAPc2 / proline on the coagulation of human plasma was measured directly in a thromboplatin time coagulation assay in rabbit brain (tissue factor, Simplastin-Excel) to initiate coagulation. Both the rNAPc2 / proline reference standard and the rNAPc2 / proline sample were diluted at 1035 nM in the assay regulator. The test instrument (Coag-A-ate® MAX, Organon Teknika, now owned by BioMérieux, Durham NC), then made a dilution set in human plasma from the start preparation and inhibited the resulting coagulation times (CTs) in seconds. The curves were defined by the linear regression adaptation CTs log of the rNAPc2 / proline against the dilution concentrations. The bioactivity of the test article was then calculated as the ratio of the curve inclination of the test article to the slope of the curve at the times of the reference standard by the activity of the reference standard. Bioburden: The Total Aerobic Count (TAC), and Yeast Count / Total Mold (TYMC), in the sample were determined by filtering two 10mL aliquots through separate 0.45 prn cellulose ester membrane filters. The filter membranes were separated and one was incubated on a TSA agar plate at a temperature of 30-35 ° C for a period of 48 to 72 hours and the other on an SDA agar plate at a temperature of 20-25 ° C. C for a period of 5 to 7 days. After the incubation period, the colony-forming units (CFU) in both types of agar were enumerated. The combined number of CFU per 100mL of sample was reported. Residual DNA: The Total DNA Threshold assay (Molecular Devices Corp. Sunnyvale CA) was specific for single-stranded DNA. It had three stages. In the reaction step, the single-stranded DNA reacted with two binding proteins in the labeling reagent. A binding protein was a high affinity single stranded DNA (SSB) binding protein of E. coli, conjugated to biotin. Streptavidin, also present, was closely linked to biotin in the SSB conjugate. The other binding protein was monoclonal anti-DNA antibody against single-stranded DNA, conjugated to the urease of the enzyme. These binding proteins formed a complex with DNA in solution at a temperature of 37 ° C. The separation stage occurred in the Threshold Workstation. The DNA complex was filtered through a nitrocellulose membrane coated with biotin. The biotin in the membrane reacted with streptavidin in DNA complex, capturing the complex. A rapid wash step removed the non-specific enzyme from the membrane. For the detection stage, the sticking (containing the microcellulose membrane coated with biotin) was placed in the Threshold Reader, which contains the substrate urea. The enzymatic reaction changed the local pH of the substrate solution. The silicon sensor recorded a change in surface potential which is proportional to the change in pH. The Threshold Workstation, the computer and the Threshold Software monitored the potential changes of the surface at each measurement site. In the measurements of these kinetics analyzed by computer, the results were quantified, using a standard curve generated previously. The Threshold Software calculated the concentration of each sample in DNA picograms. Exclusion by Size: rNAPc2 / proline was separated from other components of a mixture by size exclusion chromatography based on molecular size differences. The identity of rNAPc2 / proline was confirmed by comparing the average retention time (RT) of the duplicates of three samples with the adaptability standards of five systems. The percentage of retention times must be from 97.0% to 103.0%. The purity of the rNAPc2 / proline was calculated by diluting the rNAPc2 / proline peak water between the total peak area in the chromatogram, and expressing it as a percentage. Dilutions of the sample were prepared in cPBS at a nominal concentration of approximately 1 mg / ml and injected into a size exclusion column (Superdex 75 10/30, Amersham Biosciences). The flow range was maintained at 0.5 mL / min. The peaks were monitored by UV detection at 210 nm. Molecular Weight by Mass Spectrometry: Molecular weight was determined by electro-spraying mass spectrometry using a VG Bio-Q four-pole mass spectrometer (Quattro Upgrade) (manufactured by Micromass, Danvers, MA, currently owned by Water Corp, Bedford, MA). The sample was diluted to approximately 1 mg / mL with 0.1% aqueous trifiuoroacetic acid and injected into a Trap Cartridge previously washed for de-salary - the cartridge was then eluted through the injection port in the spectrometer. Sequence Elaboration of the Terminal-N: A sequence of the test article was elaborated through 15 residues of the N-terminal using the Procise Sequence Elaboration System of Terminal-N (Applied Biosystems, Foster City, CA). The sequence of a ß-lactoglobulin calibration standard was made through 15 residues before and after the test article. No Cys residues (cysteine) were observed in the Procise system. The sequence obtained is compared with the theoretical sequence of the test article. Example 5. Production of the rNAPc2 / proline Drug Product Example 5.1. Liquid Drug Product Manufacturing The manufacturing process of the liquid drug product of rNAPc2 / proline is underlined in Figure 5, the flow chart of the drug product. The rNAPc2 / frozen proline API was removed from storage at -20 ° C and thawed at a temperature of 2 to 8 ° C. Once thawed, the API was transferred to the preparation area of the incompuesto to combine it and mix it. To produce the drug product, the API was diluted with 65m sodium phosphate / 80mM NaCl, pH 7.0 to a final concentration of 1.0 ± 0.1 mg / mL measured by the concentration of the UV test (described above). This dilution was performed in steps with concentration measurements in the process to ensure that the specified concentration was achieved. The diluted API was then filtered through a Millipore Millipak filter of 0.2 μ? T? and placed in a short-term storage at a temperature between 2 ° C and 8 ° C. For the filling step, the diluted API was filtered in an aseptic filling suite through two Millipore Millipak filters of 0.2 μm in line. Samples were taken for the bulk sterility test. The diluted API was then filled into 2-well pre-sterilized vials which were covered and covered immediately. The objective volume of the bottles was 0.6 ml_. Prior to storage, the jars were 100% visually inspected under controlled conditions using lighting and backgrounds designed to illuminate the jars and the product, so that defective vials or jars containing visible particulates could be easily detected and removed from the batch. Then the jars were loaded onto the marked storage trays and stored in the short-term storage at a temperature of 2 ° C to 8 ° C, and long-term storage at a temperature of -20 ± 10 ° C. Table 5 is a list of the composition per vial of the liquid drug product rNAPc2 / proline.

Table 5. Drug composition of rNAPc2 / liquid proline Example 5.2. Production of the Lyophilized Drug Product A solution of the Drug Substance in the bulk of rNAPc2 / proline (in a concentration of 12 + 1.2 mg / mL in 65 mM sodium phosphate / 80 mM sodium chloride at a pH of 7.0 ± 0.1) was diluted to 3 mg / mL in a 0.2 M Alanine solution, and 25 mM monobasic sodium phosphate, pH 7.0. The rNAPc2 / diluted proline was then exchanged from regulator with the alanine / phosphate solution. The rNAPc2 / proline solution was then diluted to 2 mg / mL (measured by the UV test concentration), with an alanine / phosphate solution. 2 mg / mL of rNAPc2 / proline solution was then diluted with an equal volume of 25 mM sodium phosphate, 4% sucrose, pH 7.0, to achieve a concentration of 1.0 ± 0.1 mg / mL rNAPc2. Finally, 1 mg / mL of the formulated solution of rNAPc2 / proline was filtered using a 0.2 μm filter. For filling, 1 mg / mL of rNAPc2 / proline solution was filtered through a 0.2 μm filter. The rNAPc2 / proline was then filled into 3-cc glass jars previously sterilized and partially plugged. The bottles were then dried by freezing in a lyophilizer. After lyophilization the plugs were pushed down, and the jars were capped. The lyophilized formulation maintains high purity and sustained stability when the NAP drug product is subjected to severe temperature stresses, for example, 28 days at a temperature of 50 ° C. Table 6 is a list of the composition per vial of the lyophilized rNAPc2 / proline drug product.

Table 6. Drug composition of rNAPc2 / lyophilized proline Example 6. Prediction of Isoelectric Points of the NAP Protein The isoelectric point (pl) of several NAPs was determined to confirm that the process described herein was suitable for manufacturing other NAP drug substances and the drug substance NAP. The NAP protein sequences described in the patent 5,866,542 were calculated by the pl prediction programs. Table 7 presents the pl of the NAP proteins described in the United States patent calculated by ProtParam and Atalier Biolnformatique. ProtParam which uses ExPASy (Expert Protein Analysis System) developed by the Swiss Institute of Bioinformatics (SIB) and located at the address http://us.expasy.org/tools/protparam.html. which is received by the North Carolina Supercomputing Center (NCSS). Atalier Biolnformatique (aB¡) and is located at the address http: //www.up. univ-mrs.fr/~wabim/d abim / compo-p.html. received by Unversité Aix-Marseille I. Table 7. pl NAP Protein Prediction Name of Pl sequence calculated by ProtParam pl calculated by Atalier Biolnformatique AcaNAP5 4.32 4.10 AcaNAP6 4.25 4.03 AcaNAPc2 4.31 4.10 AcaNAPc2 / prolin 4.31 4.10 AcaNAP23 4.54 4.30 AcaNAP24 4.72 4.45 AcaNAP25 4.72 4.48 AcaNAP31.42, 46 4.28 4.07 AcaNAP44 4.74 4.48 AcaNAP48 4.34 4.13 AceNAP5 4.49 4.25 AceNAP7 4.62 4.37 AduNAP4 4.55 4.33 HpoNAP5 7.62 7.50

Claims (44)

1. CLAIMS 1. A process for the manufacture of an anticoagulant protein drug substance extracted from nematodes (NAP) which comprises: (a) a fermentation process comprising the NAP that is produced in a suitable host, wherein at least a sequence encoding the NAP is integrated into the host genome; (b) a recovery process comprising the separation of NAP from cells and cell debris; and (c) a purification process comprising the substance of the NAP drug purified from the contaminants.
2. 2. The process as described in claim 1, characterized in that the purification process further comprises introducing the NAP substance into a final drug formulation.
3. 3. The process as described in claim 2, which further comprises a filling process for producing a NAP drug product.
4. 4. The process as described in claim 1, characterized in that the NAP is selected from the group consisting of rNAPc2, rNAPc2 / proline, AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceN AP5, AceNAP7, AduNAP4, AcaNAP24 , AcaNAP25, AcaNAP44 or AcaNAP46.
5. 5. The process as described in claim 1, characterized in that the NAP is rNAPc2 / proline.
6. 6. The process as described in claim 2, characterized in that the NAP is selected from the group consisting of rNAPc2, (AcaNAPc2), rNAPc2 / proline, (AcaNAPc2 / proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44 or AcaNAP46.
7. 7. The process as described in claim 2, characterized in that the NAP is rNAPc2 / proline.
8. 8. The process as described in claim 1, characterized in that the host is Pichia pastoris.
9. The process as described in claim 1, characterized in that the fermentation process comprises a seed fermentation process wherein the host cells are cultured at a desired cell density and a production fermentation process wherein the NAP is produced to a desired title.
10. 10. The process as described in claim 9, characterized in that the production fermentation process comprises the fermentation of the glycerol batch, fermentation of the batch fed by glycerol, fermentation of methanol adaptation and fermentation of methanol induction.
11. The process as described in claim 10, which further comprises controlling the pH range for fermentation at approximately 2.9 ± 0.1 pH units during the methanol adaptation fermentation and the methanol induction fermentation.
12. 12. The process as described in claim 10, which further comprises controlling the temperature for fermentation.
13. The process as described in claim 12, which comprises maintaining the temperature of the methanol adaptation phase of the fermentation at a temperature of about 28 ± 2 ° C for approximately the first four hours and at a temperature approximately 25 + 1 ° C for the remainder of the methanol adaptation phase.
14. The process as described in claim 10, characterized in that the production fermentation is carried out for up to about seven days, during which period the NAP title continues to increase.
15. 15. The process as described in claim 1, characterized in that the recovery process comprises expanded bed ion exchange chromatography.
16. 16. The process as described in claim 15, which comprises expanded bed ion exchange chromatography of Streamiine SP XL resin.
17. The process as described in claim 1, characterized in that the purification process comprises hydrophobic interaction chromatography, collecting fractions of NAP, at least one ultrafiltration / diafiltration (UF / DF) of the NAP fractions, ion exchange chromatography and collecting the NAP fractions of the ion exchange chromatography.
18. 18. The process as described in claim 17, characterized in that the NAP fractions in the ion exchange chromatography contain the substance of the NAP drug.
19. 19. The process as described in claim 17, characterized in that the hydrophobic interaction chromatography comprises the use of hydrophobic interaction chromatography means Source 15PHE at a pH of about 3.0 ± 0.1.
20. 20. The process as described in claim 17, characterized in that the ion exchange chromatography comprises the use of Source 15Q ion chromatography media.
21. 21. The process as described in claim 17, which further comprises at least one final UF / DF of the fractions of the ion exchange chromatography.
22. 22. The process as described in claim 21, characterized in that the UF / DF exchanges the substance of the drug NAP in the regular of the final drug formulation to generate the product of the drug NAP.
23. 23. The process as described in claim 3, characterized in that the filling process comprises the bulk filtration of the drug substance NPA in the final drug formulation.
24. The process as described in claim 23, which further comprises the filling step of supplying the drug substance NAP in a dosage form to produce a NAP drug product.
25. 25. The process as described in claim 24, characterized in that the filling process further comprises freeze-drying the drug product NAP.
26. 26. A process for the manufacture of an rNAPc2 / proline drug substance which comprises: (a) a fermentation process wherein rNAPc2 / proline is produced in Plchia pastoris having at least one sequence encoding rNAPc2 / proline and is integrated into the genome, which comprises the fermentation of seed to cultivate host cells at a desired cell density and a production fermentation process comprising the fermentation of the glycerol batch, fermentation of the batch fed glycerol, fermentation adaptation of the methanol and methanol induction fermentation, for up to about seven days; (b) a recovery process comprising expanded bed ion exchange chromatography to separate rNAPc2 / proline from cells and cell debris; and (c) a purification process comprising hydrophobic interaction chromatography that utilizes hydrophobic interaction chromatography media by collecting the rNAPc2 / proline fractions, at least one uitrafiltration / diafiltration (UF / DF) of the rNAPc2 / proline fractions, ion exchange chromatography, and the collection of rNAPc2 / proline fractions from ion exchange chromatography; wherein the fractions of rN APc2 / proline from ion exchange chromatography contain the drug substance rNAPc2 / proline.
27. 27. The process as described in claim 26, which further comprises controlling the temperature for fermentation.
28. The process as described in claim 27, which comprises maintaining the temperature of the methanol adaptation fermentation at approximately 28 ± 2 ° C for approximately the first four hours and at a temperature of 25 ± 1 ° C per the rest of the methanol adaptation fermentation.
29. 29. The process as described in claim 26, which comprises maintaining the pH at about 2.9 ± 0.1 during the methanol adaptation fermentation and the methanol induction fermentation.
30. 30. The process as described in claim 26, which comprises ion exchange chromatography of the Streamiine SP XL expanded bed at a pH of about 3.2 ± 0.2.
31. 31. The process as described in claim 26, which comprises a 15PHE hydrophobic interaction chromatography at a pH of about 3.0 + 0.1.
32. 32. The process as described in claim 26, which comprises Source 15PHE ion exchange chromatography.
33. 33. The process as described in claim 26, which further comprises at least one final UF / DF of the fractions of the ion exchange chromatography.
34. 34. The process for the manufacture of a liquid rNAPc2 / proline drug product comprising the process as described in claim 26 and further comprising introducing the drug substance rNAPc2 / proline into a final drug formulation, a filling process comprising the bulk filtration of the rNAPc2 / proline drug substance in a final drug formulation, and a filling step comprising supplying the rNAPc2 / proline in a dosage form and a container to generate a product drug rNAPc2 / prollna liquid.
35. 35. The process for the manufacture of a lyophilized rNAPc2 / proline drug product, comprising the process as described in claim 34, and further comprising lyophilizing the drug or liquid product of rNAPc2 / proline in the container.
36. 36. A drug substance NAP manufactured by the process as described in claim 1.
37. 37. A drug substance NAP as described in claim 36, characterized in that the NAP is selected from rNAPc2, (AcaNAPc2) , rNAPc2 / proline, (AcaNAPc2 / proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44 or AcaNAP46.
38. 38. A NAP drug substance as described in claim 37, characterized in that the NAP is rNAPc2 / proline.
39. 39. A NAP drug product manufactured by the process as described in claim 2.
40. 40. A NAP drug product as described in claim 39, characterized in that the NAP is selected from rNAPc2, (AcaNAPc2) , rNAPc2 / proline, (AcaNAPc2 / proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44 or AcaNAP46.
41. 41. A NAP drug product as described in claim 40, wherein the NAP is rNAPc2 / pro? Ina.
42. 42. An rNAPc2 / proline drug substance as described in claim 26.
43. 43. A liquid rNAPc2 / proline drug product as described in claim 34.
44. 44. A lyophilized rNAPc2 / proline drug product as described in claim 35.